Open Veterinary Journal, (2018), Vol. 8(4): 423-431 ISSN: 2226-4485 (Print) Original Article ISSN: 2218-6050 (Online) DOI: http://dx.doi.org/10.4314/ovj.v8i4.12 _____________________________________________________________________________________________ Submitted: 09/04/2018 Accepted: 19/10/2018 Published: 15/11/2018 The prevalence of Gasterophilus intestinalis (Diptera: Oestridae) in donkeys (Equus asinus) in Egypt with special reference to larvicidal effects of neem seed oil extract (Azadirachta indica) on third stage larvae Marwa M. Attia*, Marwa M. Khalifa and Olfat A. Mahdy Department of Parasitology, Faculty of Veterinary Medicine, Cairo University, Giza, P.O. Box 12211, Egypt _____________________________________________________________________________________________ Abstract Gasterophiline larvae are of veterinary and medical importance caused specific equine intestinal myiasis. Gasterophilus intestinalis (Botfly larvae) had a wide geographical distribution. The present study explores the prevalence rate of G. intestinalis 3rd stage larvae in Egypt from January- December 2017; besides, in vitro trials to control of this larvae and evaluation of this trial using Scanning Electron Microscope (SEM) and histopathology of treated larvae. In the present study, the 3rd larval stage of G. intestinalis was found in clusters in the epithelium of the investigated stomach and infested with prevalence rate 97.2%. The highest collected numbers of larvae were found in two months; March and August (570 & 520 larvae) and lowest numbers (200 larvae) were collected in October, November, and December. The calculated LC50 and LC90 values of neem seed extract were 707.9 ppm and 1090.7 ppm. The different alteration was recorded after exposure to oil extract which showed some destruction on cuticle surface as folded and corrugated cuticle, destruction of maxillae with pits on its surface, disfigure and irregularity of cephalic spines. Histopathology of exposed G. intestinalis larvae showed different changes as thinning of cuticle at the different level (exocuticle, endocuticle, cell layers), degeneration of epithelial cells of the gut and different degree of necrosis was described. Life cycle of G.intestinalis was followed up after treatment with neem seed extract. Keywords: Donkeys, Egypt, Gasterophilus intestinalis, Larvicide, Neem seed extract. _____________________________________________________________________________________________ Introduction the highest prevalence was found in warm areas from Gasterophilus intestinalis (Diptera: Oestridae) is a Italy and Brazil (Otranto et al., 2005; Felix et al., 2007). specific obligatory parasitic myiasis larvae infesting While in Egypt Hilali et al. (1987) recorded 98.3% family equidae stomach (equine, donkeys and Zebra) infestation with G. intestinalis larvae in donkeys. mainly in the Mediterranean regions as well as U.S.A In a long time, after using of synthetic larvicides. They and China (Otranto et al., 2005; Colwell et al., 2006; contaminate the earth besides its poisonous quality to Huang et al., 2016). non-target life forms, protection improvement, and Life cycle ranged from 8-10 months which begins with dangerous impacts on human and all living wellbeing the adult female which deposits its eggs on host’s hair (Pretty, 2009). mainly forelegs. The 1st instar larvae hatch in the mouth Thus, controlling of insects and its larval stages nd rd and moult to 2 larval stage, then the L3 (3 stage) required natural alternative methods without harming remains attached to the mucosa of stomach for 8-10 the environment. Nowadays, the plant extract is a new month according to temperature and humidity (Zumpt, protocol study for controlling the disease as well as the 1965). parasite and pests. Neem seed oil has been more Invasion of G. intestinalis described by effective in controlling some parasitic disease as lice, gasterointestinal irritation including ulceration, ticks and many parasites (Okumu et al., 2007; Girish volvulus, looseness of the bowels; peritonitis may and Bhat, 2008; Giglioti et al., 2011; Abdel-Ghaffar et happen which prompts intestinal break (Otranto et al., al., 2012; Ruchi et al., 2014). 2005). There are numerous reports of human myiasis The previous literature reveals that still no work had caused by Gasterophilus spp. larvae causing been done on this Egyptian neem seed extract by the ophthalmo-myiasis (Anderson, 2006; Yang et al., scientist on equine botfly larvae. Along with this lines, 2013). the present study explores the prevalence rate of G. Gasterophilus intestinalis had a wide geographical intestinalis 3rd stage larvae in Egypt from January- distribution in different countries as Morocco, Europe, December 2017; besides, in vitro trials to control of this Italy, Turkey and Saudi Arabia (Pandey et al., 1992; larvae and evaluation of this trial using Scanning Colebrook and Wall, 2004; Otranto et al., 2005; Electron Microscope (SEM) and histopathology of Gökcen et al., 2008 ; Al Anazi and Alyousif, 2011) but treated larvae. ________________________________________________________________________________________________________ *Corresponding Author: Marwa Mohamed Attia. Department of Parasitology, Faculty of Veterinary Medicine, Cairo University, Egypt. Fax: +202-35725240; +202-35710305. Email: [email protected] 423 http://www.openveterinaryjournal.com M.M. Attia et al. Open Veterinary Journal, (2018), Vol. 8(4): 423-431 ________________________________________________________________________________________________________ Materials and Methods ppm, 300 ppm, 500 ppm, 700 ppm, 900 ppm and 1000 Collection of Gasterophilus intestinalis larvae ppm) which were freshly prepared in distilled water A total of 144 stomach donkeys (12 stomach /month) with few drops of Tween 80 as an emulsifier. Fifty were inspected during a period from January-December fresh, actively movement G. intestinalis L3 were used 2017 from the slaughtered house in Giza, Zoo abattoir in each test. Each group of larvae was placed in 100 ml (Giza, Egypt) for detection of infestation of solution of each concentration, immersed for 10 Gasterophilus spp. 3rd stage larvae. The donkeys were minutes and then the solution was continuously stirred collected from three governorates Giza (40), Fayoum during the test; Each concentration was tested in two (48) and Beni Sweif (56). The governorates were replicates; each replicate was 50 larvae (Smith et al., located south of Cairo city at a distance of 2.5, 103 2000; Khater et al., 2009, 2013). and119 Km., respectively. Each stomach was examined The negative control groups (non - exposed groups) of with naked eyes and Gasterophilus larvae were larvae was kept in (group with distilled water alone collected from each infested donkey and counted then and group in distilled water with few drops of Tween placed in a separate vial containing the saline solution 80). Then, treated larvae with neem oil were removed (0.9% NaCl, GmbH, Germany). All larvae were from oil and kept in plastic cups with filter papers examined on the same day of collection at the (Whatman No. 1) and kept at 27 ± 2 °C and 80±5 % Parasitology Department, Faculty of Veterinary relative humidity (RH). The mortality of larvae in all Medicine, Cairo University, Giza, Egypt for further cups was followed up in the same day of treatment. studies. This study was approved from The Animal Larvae were considered alive or dead according to Ethical Committee of Faculty of Veterinary Medicine, larval motility (Khater and Ramadan, 2007; Khater et Cairo University number: CU/II/F/18/104. al., 2013). The Collected larvae Larval mortality was determined daily until pupation All larvae subjected to two processes; identification occurred. The larvae were incubated at 32∘c and 80-85% study and control one. Therefore,10 larvae from each RH. Then each concentration was divided into four governorate were identified using a stereoscopic beakers (each beaker had 25 larvae) containing a sterile microscope (LEICA M60, United states) then sand and covered with gauze. The beakers were put in a (scanning electron microscope) study. Other fresh large dissector containing a suitable amount of salt active instar larvae were used in control study using solution to adjust the humidity. The larvae examined neem seed oil. daily for any mortality and pupal formation. The Morphological identification of 3rd instar larvae developed pupae, at each concentration, were counted For SEM, ten larvae were collected. They were and placed in separate cages until adults emergence. prepared by serial washing in saline solution and fixed Assessment of larvicidal efficacy of neem oil extract in 2.5% glutaraldehyde as previously described by Scanning electron microscope (SEM) study Colwell et al. (2007), Hilali et al. (2015) and Attia Five larvae were chosen from the last two (2018). Larvae were dehydrated through passage in concentrations (900 ppm and 1000 ppm) and five from ascending ethanol degrees, which dried in Co2 critical control group (non treated) prepared by serial washing drier (Autosamdri-815, Germany) and glued over in saline solution and fixed in 2.5% glutaraldehyde as stubs; coated with gold in a sputter coater (Spi-Module described by Hilali et al. (2015) and Attia (2018). sputter Coater, UK). Specimens were photographed Specimens were processed as previously described. using SEM (JSM 5200, Electron prob Microanalyzer, Histopathological studies Jeol, Japan) at Faculty of Agriculture, Cairo University, Larvae were taken from those